Camp-responsive transcriptional enhancer binding protein

ABSTRACT

This invention is directed toward the characterization and cloning of a cAMP-responsive transcription enhancer binding protein (CREB). This protein, CREB, is a transcriptional activator which activates transcription in eukaryotic cells. This CREB protein can be used to increase or decrease production of proteins by stimulating expression of a recombinant gene that is operably-linked to the CRE enhancer element and responsive to cAMP.

The research underlying this patent application was supported by National Institutes of Health Grant DK-25532; the government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US89/05234, filed Nov. 20, 1989 and is a continuation-in-part of U.S. application Ser. No. 07/272,980, filed Nov. 18, 1998, now abandoned, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

This invention is in the field of genetic engineering, specifically directed toward the characterization and cloning of a cAMP-responsive transcription enhancer binding protein (CREB). The invention is also directed to methods for the use of the CREB protein to increase or decrease the production of specific proteins in eukaryotic cells by activating transcription of a recombinant gene in response to cAMP.

BACKGROUND OF THE INVENTION

Within the cell, transcriptional selectivity of eukaryotic genes is mediated by complex control regions composed of different combinations of promoter and enhancer elements. These regions are arrayed in tandem to allow multiple distinct regulatory factors to function coordinately to potentiate RNA synthesis. This mosaic arrangement of eukaryotic transcriptional regulatory elements provides different genes with the possibility of utilizing some of the same regulatory elements.

Enhancers are sequence-specific DNA transcriptional regulatory elements that function in cis to stimulate the transcription of genes placed in proximity to them. Generally, elements that function in cis are recognition sites for cellular proteins (Dynan, W. S. et al., Nature 316:774-778 (1985)). The cellular proteins which recognize enhancer sequences are often expressed in a manner which is tissue-specific or species-specific, or dependent upon the hormonal environment. Upon binding of the appropriate protein to the enhancer region, transcription of genes under the control of, that is, operably-linked to the enhancer is facilitated, resulting in an increased transcriptional expression of the gene, and thus in an increased expression of any protein for which the gene codes.

Enhancers are not orientation dependent elements like promoter regions are. Enhancer sequences can be oriented in either direction relative to the direction of transcription of the operably-linked gene. In addition, the sequence itself may be located anywhere in the general area of the gene, such as 5' to the promoter region, 3' to the transcriptional termination site, or even within a transcribed region of the gene, for example, in an intron. A gene may be under the transcriptional regulatory influence of multiple copies of the same enhancer, or the gene may be under the transcriptional regulatory influence of a group of different enhancers, each enhancer in the group conferring a different regulatory response on the operably-linked gene. Examples of these responses include an ability to transcriptionally respond to different agents or hormones, and tissue-specific expression of the gene.

Because of their relative orientation independence, enhancers can be located at varying distances from the promoter and transcription unit of the gene and yet still be operably-linked to that gene. The transcription unit is that sequence of a gene which is transcribed. The distance will vary with the transcriptional strength of the promoter and enhancer. Typically, on the average, enhancers are located within 200 bases upstream from the promoter site which itself determines the base at which transcription begins.

Cyclic adenosine monophosphate (cAMP) is the intracellular second messenger for many hormones or biological mediators and is known to be active in the regulation of gene expression in both prokaryotes and eukaryotes. In eukaryotes, the regulation of transcription by cAMP has been extensively studied in animals and tissue culture cells. Increasing the intracellular cAMP concentration with hormones such as glucagon or other agents such as cAMP analogs or beta-adrenergic agonists induces the transcription of many genes in a tissue-specific manner, including somatostatin (Montminy, M. R. et al., Proc. Natl. Acad. Sci. USA 83:6682 (1986)), the alpha subunit of human chorionic gonadotropin (Silver, B. J. et al., Proc. Natl. Acad. Sci. USA 84:2198 (1987); Jameson, J. L. et al., Endocrinology 119:2570 (1986); Delegeane, A. M. et al., Mol. Cell. Biol. 7:3994 (1987); Jameson, J. L. et al., Mol. and Cell. Biol. 7:3032 (1987); Deutsch, P. J. et al., Bio. Chem. 262:12169 (1987)); phosphoenolpyruvate carboxykinase (Short, J. M. et al., Biol. Chem. 261:9721-9726 (1986)), tyrosine hydroxylase (Lewis, E. J. et al., Proc. Natl. Acad. Sci. USA 84:3550-3554 (1987)), and c-fos (Greenberg, M. E. et al., J. Biol. Chem. 160:14101-14110 (1985)).

Cyclic AMP-responsive genes contain a sequence homologous to the sequence TGACGTCA located on the 5' side of their mRNA cap sites. This sequence has been termed a cAMP-responsive enhancer element (CRE). Deletion mutagenesis of cAMP-inducible genes has shown that the cAMP-responsive enhancer element is contained within a domain necessary for cAMP-mediated induction of transcription.

Similar consensus DNA regulatory elements involved in the stimulation of gene transcription have been identified for other molecules, such as for the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) (Imbra, R. J. et al., Mol. and Cell. Bio. 7:1358 (1987); Angel, P. et al., Cell 49:729 (1987); Tsukada, T. et al., Bio. Chem. 262:8743 (1987); Angel, P. et al., Mol. and Cell. Biol. 6:1760 (1986); Chiu, R. et al., Nature 329:648 (1987); Angel s P. et al. Mol. and Cell. Biol. 74:2256 (1987); Comb, M. et al., Nature 323:353 (1986)). However, notably, the sequence of the octameric cAMP-response element, CRE, (5'-TGACGTCA-3') differs from that of the heptameric TPA-response element, TRE, (5'-TGAGTCA-3') by a single base.

Early studies suggested that transcriptional stimulation by both cAMP and TPA was mediated through a common DNA sequence present in the 5' regulatory region of the enkephalin gene, 5'-TGCGTCA-3'(Comb, M. et al., Nature 323:353 (1986)). However, a DNA binding protein of 47 Kd (AP-1 or c-jun) was isolated and shown to mediate TPA but not cAMP induction of SV40 gene transcription through a mechanism involving sequence-specific binding to the TRE motif (Lee, W. et al., Cell 49:741 (1987)). Similarly, a 43 Kd protein termed CRE-binding protein (CREB) has been identified that binds to a CRE sequence in the 5' regulatory region of the rat somatostatin gene (Montminy, M. R. et al., Nature 328:175 (1987)). In placental JEG-3 cells, a 38 Kd protein was shown to bind to CRE (Deutsch, P. J., et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988)). However, the sequence of CREB had not previously been determined, precluding the undertaking of detailed structural or functional studies.

Anti-sense RNA refers to RNA synthesized with a sequence complementary to that found in a specific mRNA. Anti-sense RNA has been used to inhibit, in a specific manner, the expression of the protein whose mRNA is being hybridized by the anti-sense RNA. Inhibition by hybridization in eukaryotes is thought to occur at the level of processing of the mRNA (thus preventing its translocation to the cytoplasm) while in prokaryotes it is thought to occur at translation of the mRNA. At either step, the ultimate result is to effectively stop expression of the target protein whether the system is bacteria, plants or other eukaryotic systems (Knecht, D. A. et al., Science 236:1081-1086 (1987); Van Der Krol, A. R. et al., Nature 333:866-869 (1988); Cabrera, C. V. et al., Cell 50:659-663 (1987); Boulay, J. L. et al., Nature 330:395-398 (1987); Rothstein, S. J. et al;, Proc. Natl. Acad. Sci. USA 84:8439-8443 (1987); Ecker, J. R. et al., Proc. Natl. Acad. Sci. USA 83:5372-5376 (1986); Lichtenstein, D., Nature 333:801-802 (1988)). However, it has not previously been known to use cAMP with anti-sense RNA technology to control the expression of specific proteins in a manner capable of acute regulation in response to the levels of cAMP in the system.

SUMMARY OF THE INVENTION

This invention is directed toward the characterization and cloning of DNA encoding a cAMP-responsive transcription enhancer binding protein (CREB) and the recombinant protein produced by this DNA. This protein, CREB, is a DNA binding protein and is capable of recognizing and binding to DNA containing the cAMP enhancer element, CRE, and selectively activating transcription of genes operably-linked to the enhancer element in eukaryotic cells. The present invention also provides methods for the selective stimulation of transcription of recombinant genes using the CREB protein. Especially, the present invention provides methods for the selective stimulation of transcription of recombinant genes using the CREB protein in response to cAMP. The present invention further provides methods for the selective inhibition of protein expression using the CREB protein of the invention and cAMP to stimulate the synthesis of an anti-sense RNA. The methods of the invention allow, for the first time, the acute regulation of specific protein levels, in both a positive and negative manner using cAMP or hormones or other agents which act through cAMP to enhance transcription.

DESCRIPTION OF THE FIGURES

FIG. 1. Primary structure of CREB.

The basic region and leucine zipper sequence located at the carboxyl terminus of the protein are underlined. The periodic array of leucine residues (circled) spaced seven residues apart would form the hypothetical alpha helix involved in protein-protein contacts (Landschultz, W. H. et al., Science 240:1760 (1988)). Preliminary evidence indicates that the methionine at a position one is the translational start site in vivo. Amino acid sequence is in single letter code.

FIG. 2. Diagram of the hypothetical functional domains of CREB.

Basic region and leucine zipper sequence at the carboxyl terminus provide the putative DNA binding domain. The amino terminal residues 1-268 constitute the proposed negatively charged activation domain in which 25 of the 36 charged amino acids (exclusive of the two histidine residues) are glutamic and aspartic acids. This region of the protein has characteristics of a "negative noodle" hypothesized to be involved in the coupling of DNA binding proteins to other transcriptional factors (Sigler, P. S., Nature 333:210 (1988); Hope, I. A. et al., Nature 333:635 (1988); Ma, J. et al., Cell 48:847 (1987); and Gill, G. et al., Cell 51:121 (1987)).

FIG. 3. Comparisons of leucine zipper regions in the structure of CREB and other DNA binding proteins.

Alignment of leucine zipper regions of CREB and several other proteins. Leucines reside at every seventh position, a periodicity required for hypothetical alignment of the leucines on the same spoke of an idealized alpha helix.

FIG. 4. Comparison of sequence similarities between CREB and c-jun.

A region of primary sequence similarity between CREB and c-jun is localized to the basic region that is adjacent to the leucine zipper region. Boxed residues are shared by the two DNA binding proteins. Arginine and lysine are considered interchangeable. Arrows point to leucines in the zipper region. Sequence positions numbered correspond to those of CREB, FIG. 2, and c-jun (Bohmann, D. et al., Science 238:1386 (1987); and Angel, P. et al., Nature 332:166 (1988)).

FIG. 5. Secondary structure comparisons of CREB and c-jun.

The plots compare four parameters of secondary structures: alpha helix (A), beta strand (B), random coil (C), beta turn (T), as well as hydrophobicities (H) (Garnier, J. et al., J. Mol. Biol. 120:97 (1978); and Kyte, J. et al., J. Mol. Biol. 157:105 (1982)) (MacGene Plus computer program). Numbers at top refer to the sequence of CREB (326 residues) and c-jun (331 residues). Note overall similarities in the secondary structures of the two proteins despite notable absence of similarities of the primary amino acid sequence as seen in FIG. 1 between CREB and c-jun as cited in Bohmann, D., et al., Science 238:1386 (1987) and Angel, P., et al., Nature 332:166 (1988).

DEFINITIONS

In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

Selectively activate transcription. To "selectively activate transcription" means to activate or increase the transcription of a heterologous gene or group of genes, without activating transcription in general.

Selectively inhibit expression. To "selectively inhibit expression of a protein means to inhibit, decrease or stop the expression, transcription, mRNA processing, translation or synthesis of a specific protein or group of proteins, either endogenous or heterologous, without inhibiting the ability of the cell to express, transcribe, process, translate or synthesize proteins in general.

DNA element. A "DNA element" is a DNA sequence which confers a unique property on a gene which is operably-linked to it. DNA elements include enhancer sequences and may confer hormonal responsiveness or tissue-specific expression on a gene.

Minimal selectable region. The term "minimal selectable region" refers to an isolatable DNA region or sequence containing the sequence information required to confer a unique function or other property on a DNA construct which contains the minimal selectable region. Examples of minimal selectable regions are a promoter sequence, the CREB sequence, the CRE enhancer element, a heterologous gene, transcriptional stop sites, and the like.

Operably-linked. By "operably-linked" is meant that a DNA element or minimal selectable region is located at a site which places a gene or group of genes under the control or influence of that element or region. For example, an operably-linked promoter sequence is the promoter for the gene; an operably-linked enhancer sequence is capable of enhancing the transcription of genes operably-linked to it.

DETAILED DESCRIPTION OF THE INVENTION

Cyclic AMP is an intracellular second messenger that activates transcription of many cellular genes. A cAMP-responsive transcriptional element (CRE) has been identified as a palindromic consensus DNA sequence, TGACGTCA. This sequence functions as a DNA enhancer specific for cAMP regulatory events. Although the CRE is a component of the regulatory region of cAMP-responsive genes, the presence of this sequence is not itself sufficient for cAMP inducibility. Exposure of the cell to stimuli that increase cAMP is necessary to stimulate a cascade of events which ultimately produces a transcriptionally active (or activated) complex between the CRE element and a specific transcriptional factor which binds to this element. According to this invention, the transcriptional factor has now been characterized and cloned. This transcriptional factor is a unique CRE-binding protein, abbreviated CREB. CREB is a DNA binding protein which specifically responds to cAMP-induced regulatory events by binding DNA that contains the CRE enhancer element and stimulating transcription. The CREB protein of the invention can be used to regulate the transcription of recombinant genes that have been operably-linked to the CRE enhancer. Such constructs can be used to increase or decrease the expression of specific proteins in a cAMP-dependent manner.

The DNA encoding CREB of the invention was found by screening a placental λ gt11 library for expression of specific CRE-recognition and binding proteins using the CRE sequence as a radioactive probe. A cDNA encoding a protein of 326 amino acids with the binding properties of a specific CRE-recognition and binding protein (CREB) was isolated. The CREB protein encoded by the isolated DNA contains a carboxy terminal basic region adjacent to a leucine zipper sequence which is similar to sequences believed to be involved in DNA binding and in protein-protein contacts in several other DNA-associated transcriptional proteins, including c-myc, c-fos, c-jun and GCN4. CREB also contains an amino terminal acidic region proposed to be a potential transcriptional activation domain. The putative DNA binding domain of CREB is structurally similar to the corresponding domains in the phorbol ester-responsive proto-oncogene c-jun and the yeast transcription factor GCN4 that bind to a heptameric DNA element, TGAGTCA, closely related to the CRE octamer.

Based upon the deduced protein sequence of this cloned cDNA, the cDNA encodes a full-length CREB protein with a calculated molecular mass of 35,024 daltons. This conclusion is consistent with the finding of a 38 Kd CREB protein present in extracts of JEG-3. human choriocarcinoma cells, assuming that the cellular protein is post-translationally modified. The apparent discrepancy in molecular weights between this human placental CREB of 38 Kd and the 43 Kd CREB identified in rat adrenal cells (PC-12) by Montminy and Bilzikjian (Montminy, M. R. et al., Nature 328:175 (1987)) could be due to species-specific differences in primary structure, post-translational modifications, or the existence of multiple CREB proteins which are part of a larger family of CREB transcriptional activators. Recent reports have suggested that a 45 Kd E1A-regulated cellular transcription factor (ATF) is similar or identical to CREB and that ATF/CREB can be regulated in vivo by both the adenovirus E1A protein and cAMP (Lin, Y-S. et al., Proc. Natl. Acad. Sci. USA 85:3396 (1988); and Hardy, S. et al., Proc. Natl. Acad. Sci. USA 85:4171 (1988)).

Isolation of the cDNA encoding CREB will facilitate studies aimed at addressing the basis for the molecular heterogeneity of CREB and CREB-like proteins and the interactions of CREB-like, fos-related, and jun-related proteins in the transcriptional activation of genes.

In addition a recombinant source of CREB will greatly facilitate studies directed towards elucidating the mechanisms through which cAMP modulates intracellular metabolism by directing transcriptional events. Genes suspected of being under cAMP control can be evaluated in terms of their ability to respond to, or bind, the CREB of the invention. Recombinant CREB will also facilitate studies directed towards elucidating the transcriptional mechanism-of-action of hormones and other agents suspected of acting through cAMP by examining their ability to influence CREB-directed transcription.

Further, since CREB is a transcriptional activator which activates transcription of genes operably-linked to the CRE element in eukaryotic cells, according to the methods of this invention, CREB can be used in conjunction with CRE and especially with cAMP to increase production of heterologous proteins and polypeptides by stimulating expression of recombinant genes. The CREB protein of the invention can also be used to activate the transcription of an RNA sequence which is not translated, such as an RNA sequence complementary to a known mRNA, or anti-sense RNA. Expression of an anti-sense RNA can be used to block the expression of endogenous or heterologous proteins.

Lastly, the CREB-CRE transcription methods of the invention provide methods of cAMP-controlled mutagenesis in eukaryotic cells.

Thus, the invention encompasses any construct or set of constructs which relies on CREB and CRE recognition or binding to alter the expression of a homologous or heterologous gene product by enhancing the transcription of a recombinant RNA.

The preferred hosts are mammalian cells, grown in vitro in tissue culture, or in vivo in animals. Mammalian cells provide post translational modifications to proteins and polypeptides including correct folding or glycosylation at correct sites.

Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or Chinese hamster ovary CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/O-AG14 or the myeloma P3×63Sg8, and their derivatives. Preferred mammalian host cells include SP2/O and J558L. In one preferred embodiment the CREB sequence of the invention is provided to the host cell in a transcribable and translatable minimal selectable region on the same vector construct as that providing the CRE minimal selectable region operably-linked to a recombinant gene. In another preferred embodiment, the CREB sequence of the invention is provided to the host cell in a transcribable and translatable minimal selectable region on a vector construct which is separate and maintained as a separate replicating unit from that providing the CRE minimal selectable region operably-linked to a recombinant gene.

The CREB-encoding DNA of the invention, in an expressible form, can also be inserted into the chromosome of the host cell. CREB functions in trans which means that it is the diffusible product of the CREB gene which functionally activates expression of genes operably-linked to the CRE element in response to cAMP. Therefore, it is necessary only that the minimal selectable region bearing the CREB gene of the invention be present in the same cell as the minimal selectable region providing the CRE element; the CREB DNA sequence need not be physically linked to the plasmid or element bearing the CRE sequence.

The CREB protein as depicted in FIG. 1, or active CRE recognition and binding fragments thereof, may be used in the method of this invention in several embodiments. It is to be understood that while the full octameric CRE sequence is necessary to the construct, it is not necessary that the full-length CREB sequence be used. Only the portion of the CREB sequence necessary to functionally activate transcription and recognize and bind to DNA containing the CRE sequence is needed. Active CRE recognition and binding fragments may be determined by routine screening. Further, FIG. 2 provides a diagram of the proposed functional domains of CREB.

It is also to be understood that by using techniques known to those of ordinary skill in the art it is possible to design chimeric constructs of the CREB protein which contain the ability to recognize the CRE element and thus respond to cAMP in a highly specific manner but which bind to or activate different targets in DNA. Such a chimeric construct might ligate the amino-terminal portion of the CREB protein of the invention with the DNA binding and "zipper" region from another DNA binding protein, or, place the DNA binding and zipper portion of the CREB protein with an alternate amino-terminal domain thus altering the transcriptional targets of the cAMP response.

The promoter chosen to regulate expression of the CREB protein of the invention may be the same or different from the promoter chosen to regulate the recombinant gene. In one embodiment, no enhancer is operably-linked to the promoter operably-linked to CREB. In a preferred embodiment, the CRE element is operably-linked to the CREB promoter so that CREB synthesis enhances its own transcription and expression. In another embodiment, enhancers conferring tissue or species specificity, such as GCN4 in yeast, are operably-linked to the CREB promoter, which may or may not be operably-linked to CRE also. Any promoter capable of directing the RNA polymerase II transcription of the operably-linked recombinant CREB gene is applicable to the methods of the invention. RNA polymerase II is that RNA polymerase which specifically transcribes DNA into mRNA. Promoter selection is important only in that it allows the host cell to express enough of the CREB protein of the invention so that the level of CREB protein is not a factor limiting the stimulation of the CRE-recombinant gene construct.

In one embodiment the promoter used for the CREB construct of the invention is the homologous CREB promoter from the human placenta. In another embodiment, the CREB promoter from the tissue or cell line of interest is used. Because CREB should not be in limiting quantities it is desirable that a strong promoter be used. By strong promoter is meant a promoter possessing a high affinity for RNA polymerase, as one which provides an accessible RNA polymerase entry site. Examples of strong eukaryotic promoters include promoters from SV40, actin, Rous sarcoma virus, herpes virus, thymidine kinase, and adenovirus MLTV.

The CREB construct as shown in FIG. 1 provides the translational stop and start sites and capping site necessary for the proper translation of the sequence into a functional CREB protein in eukaryotic systems.

For a mammalian host, several possible vector systems are available for the expression of either or both the CREB protein of the invention and the heterologous recombinant protein. One class of vectors utilizes DNA elements which provide autonomously replicating extra-chromosomal plasmids, derived from animal viruses such as bovine papilloma virus, polyomavirus, adenovirus, or SV40 virus. A second class of vectors relies upon the integration of the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g. antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Preferably the marker is a dominant-acting marker which produces a discernable change in the phenotype of normal cells. Colbere-Garapin, F. et al., J. Mol. Biol. 150:1 (1980).

The constructs may be introduced into a host cell by transformation in conjunction with a gene allowing for selection where the construct will become integrated into the host genome. Usually the construct will be part of a vector having a replication system recognized by the host cell. In another embodiment of this invention, the host cell has been modified prior to transformation with the construct containing the CRE and the heterologous gene so that the cell is already actively expressing the CREB protein, or active CRE recognition and binding fragments, or, maintains the CREB protein or active CRE recognition and binding fragment integrated in its genome.

When the CREB-encoding DNA of the invention is inserted into the host cell chromosome, DNA amplification techniques can be used to increase the copy number of the CREB gene. Amplification serves the same purpose as a multi-copy plasmid in so far as it results in multiple copies of a functional gene.

Another preferred host is yeast. Yeast provide substantial advantages in that yeast are capable of post-translational peptide modifications including glycosylation (Kukuruzinaka, M. A. et al., Ann. Rev. Biochem. 56:915-944 (1987)), and a number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number plasmids which promote the production of large amounts of the desired protein. Yeast also recognize leader sequences on cloned mammalian gene products, and can secrete peptides bearing leader sequences (i.e., pre-peptides). Botstein, D. et al., Science 240:1439-1443 (1988); Struhl, K., Nature 305:391-397 (1983); Sherman, F. et al., Methods in Yeast Genetics-Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1983.

Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed yeast genes coding for proteins, especially glycolytic enzymes such as phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, glucokinase, hexokinase, pyruvate kinase, pyruvate decarboxylate, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, triosephosphate isomerase, phosphoglucose isomerase, alcohol dehydrogenase, isocytochrome C and the like, produced in large quantities when yeast are grown in medium rich in glucose can be utilized. See, for example, Broach, J. R., Meth. Enz. 101:307 (1983); Stinchcomb et al., Nature 282:39 (1979); Tschempe et al., Gene 10:157 (1980); and Clark, L., et al., Meth. Enz. 101:300 (1983). Known glycolytic genes can also provide very efficient transcription control signals. Hess et al., J. Adv. Enzyme Reg. 7:149 (1968); Hitzeman et al., J. Biol. Chem. 255:2073 (1980); and Holland, M. J., J. Biol. Chem. 256:1385 (1981).

Another preferred host is insect cells, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used. Rubin, G. M., Science 240:1453-1459 (1988). Alternatively, baculovirus vectors can be engineered to express large amounts of protein in insects (Jasny, B. R., Science 238:1653 (1987); Miller, D. W., et al., in Genetic Engineering (1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

To express a heterologous protein in the method according to this invention, transcriptional and translational eukaryotic signals recognized by the eukaryotic host are necessary. Expression vehicles for production of heterologous protein include plasmids or other vectors as described for the CREB protein of the invention. The vector chosen to carry the CREB minimal selectable region and the vector chosen to carry the minimal selectable region containing the CRE element operably-linked to a heterologous recombinant gene, must also contain replicon and control sequences which are derived from species compatible with the host cell and used in connection with the host. The vector ordinarily carries a replicon site, as well as specific genes which are capable of providing phenotypic selection in transformed cells.

The DNA sequence coding for the heterologous protein may be obtained in association with its homologous promoter region from genomic DNA. To the extent that the host cells recognize the transcriptional and translational regulatory signals and the mRNA processing signals associated with the heterologous protein's gene, then the regions 5' or 3' to the heterologous protein's transcribed coding sequence and the introns may be retained and employed for transcriptional and translational processing and regulation.

In another embodiment the minimal selectable region containing the recombinant gene construct operably links a homologous promoter region for the recombinant gene or a heterologous promoter to a recombinant gene containing no introns.

According to the methods of the invention, stimulation of transcription in response to cAMP can be used in combination with other transcriptional and translational regulatory sequences. Other transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, etc., may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.

The contiguous non-coding region 5' to the heterologous protein which is retained after processing the introns out of the mRNA precursor will normally include those sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. Usually the 5'-non-coding sequence will be at least 150 bp, more usually at least 200 bp, usually not exceeding about 2 kbp, more usually not exceeding about 1 kbp.

The non-coding region 3' to the heterologous protein coding sequence in the native gene may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3'-region naturally contiguous to the DNA sequence coding for the translated region, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' untranslated region functional in the host cell may be substituted with the 3' region of a highly transcribed protein. In this method, the choice of protein for the substituted 3' region would depend on the cell system chosen for production.

The construct for the heterologous protein will comprise the CRE sequence. CRE is a palindrome, which is a bilaterally symmetrical DNA sequence which, therefore, reads the same in both directions. In the methods according to this invention, to express a protein, a construct is made which contains minimal selectable regions comprising a CRE element operably-linked to a promoter which is operably-linked to a heterologous gene. The orientation of the CRE sequence of the invention can be either 5' or 3' relative to the direction of transcription of the recombinant gene. The CRE element may be located either 5' to 3' to, or within the transcriptional unit itself. By transcriptional unit is meant the DNA sequence that is transcribed into RNA.

More than one CRE sequence may be inserted into the construct and operably-linked to the promoter of the heterologous gene if the addition of additional CRE elements does not detrimentally alter the ability of cAMP to stimulate transcription of the gene. In addition, CRE elements may be separated by DNA spacers of variable length and sequences so long as those spacer regions are not detrimental to the ability of the CREB protein to recognize, bind and stimulate the transcription of the heterologous gene.

Once the vectors or minimal selectable regions containing the constructs have been prepared for expression, they may be introduced into the appropriate host. Various techniques may be employed to transform the host with the vectors or constructs, such as protoplast fusion, calcium phosphate-precipitation, electroporation, viral infection or other conventional techniques. After the transformation or transfection, the cells are grown in a selective medium, where untransformed cells are killed, leaving only cells transformed with the constructs of the invention.

Expression of the heterologous gene(s) is stimulated by the addition of cAMP or, by the addition of any analog or hormone acting through cAMP to which the cell is responsive, directly to the culture medium or animal containing the host cell. Cell-membrane permeable, stable analogues of cAMP such as 8-Bromo-cAMP or dibutyryl cAMP may be used. Alternatively, the turpene compound forskolin can be used to stimulate the enzyme adenylate cyclase within the cell, thereby resulting in the cellular synthesis of cAMP.

In a preferred embodiment, 0.1-5 mM 8-Bromo-cAMP or dibutyryl cAMP is used in the method of the invention to stimulate transcription. Alternatively, any concentration of cAMP or an active derivative thereof may be used. The concentration which is required is limited only by the ability of that concentration to effectively induce the desired transcriptional response.

In addition to direct addition of cAMP or an active derivative thereof to the host cell or animal or medium containing the host cell, any hormone or other agent which is able to increase levels of cAMP in the host cell may be used, such as glucagon or β-adrenergic agents. The hormone or agent is limited only by the ability of the cell to respond to the hormone or agent in a cAMP dependent manner.

Although the exact mechanism of the regulatory steps are not known, it is believed that the presence of cAMP may influence the synthesis, activity, recognition ability and/or binding affinity of the CREB protein, which in turn, binds to the CRE palindrome, signalling the expression of the heterologous or recombinant gene.

The expressed heterologous protein or polypeptide may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.

Accordingly, it is within the scope of the methods of the present invention to stimulate the transcription of a heterologous translatable mRNA by cAMP where the increased levels of the heterologous, translatable mRNA results in an enhanced expression of a heterologous protein.

It is also within the methods of the invention to stimulate the synthesis of a heterologous but non-translatable RNA.

Accordingly, the recombinant gene may comprise any regulatory RNA sequence capable of being transcribed under direction of a CRE-regulatable promoter, but not able to be translated. By regulatory RNA is meant an RNA sequence capable of infuencing the transcription, processing or translation of another RNA sequence. In a preferred embodiment, the transcribed recombinant RNA sequence is an anti-sense RNA; that is, it is complementary to, and capable of hybridizing with, a known mRNA. According to the methods of the invention, upon the induction of the transcription of an anti-sense RNA under the direction of the cAMP-regulatable CREB-CRE recognition and binding, expression of the protein for which a mRNA codes would decrease or stop due to hybridization of the anti-sense strand of RNA with the sense strand. Accordingly, levels of the protein for which the mRNA codes fall. The mRNA Whose processing or translation is being inhibited by hybridization to the anti-sense RNA may be homologous to the host cell or heterologous to it. The method of the invention is especially applicable to the insertion of the minimal selectable region containing the CRE element operably-linked to a promoter directing the transcription of an anti-sense RNA sequence into the genome of the host cell, in a manner which allows it, in a cAMP dependent manner, to inhibit the over-expression of a protein detrimental to the viability of the cell. Such expression may utilize the CREB protein of the invention or the host's endogenous CREB protein.

The methods of the invention are also adaptable as methods of in vivo mutagenesis. For example, in yeast, by encoding a transposase in the cAMP- regulatable recombinant gene, transposition-dependent DNA mutational events may be placed under the control of cAMP. Cells exhibiting the desired mutant phenotype could then be isolated and characterized.

Alternatively the methods of the invention may be used as a method of mutagenesis which examines function of a protein by using the methods of the invention not to alter the genotype itself, but to effectively create cells deficient in a protein in response to a cAMP-directed transcription of an anti-sense RNA.

The advantage of the methods of the invention include their ability to provide reversible, acute methods of target-specific control of RNA expression or protein expression. The effect of the methods of the invention are reversible by decreasing, removing or metabolizing the levels of cAMP in the medium or cell. That is, by merely manipulating the levels of cAMP in the host cell for a desired period of time, expression of the gene operably-linked to the CRE element is controlled. In addition, the methods are acute because they are rapid and do not depend on the ability of the cell to replicate.

Having now generally described this invention, the same will be better understood by reference to specific examples, which are included herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES

A screening technique recently described by Singh and co-workers (Singh, H. et al., Cell 52:415 (1988)) was used to isolate a cDNA encoding an expressed protein that binds specifically to the CRE recognition site.

A primary screening of a human placental expression library with a radioactive synthetic CRE duplex probe yielded 23 positive recombinant phage plaques. After plaque purification through four successive screenings, only five positive clones remained. A recombinant phage that did not bind the radioactive probe was also plaque purified as a negative control. To establish the specificity of the binding of the radioactive probes, an array of synthetic oligonucleotide duplexes for which transcriptional activities and protein-binding characteristics have been elucidated in detail was utilized (Deutsch, P. J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988); Deutsch, P. J. et al., J. Bio. Chem. (in press)). These duplex DNAs fall into three groups. The "active CRE's" consisted of the CRE octamer element flanked by several bases as they occur in the cAMP responsive chorionic gonadotropin α subunit and somatostatin genes and the collagenase gene in which the TRE heptamer was converted to a transcriptionally active CRE octamer (Deutsch, P. J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988)). These sequences could impart transcriptional stimulation in response to 8-bromo-cAMP, when linked to a minimal promoter element, and could successfully compete for binding to a labeled "active CRE" in gel-shift assays. The corresponding TRE's produced band-shift patterns different from those of the CRE's and could not compete for binding to a labeled "active CRE." Finally, the "inactive CRE's" consisted of the CRE octamer in the contexts of the surrounding bases of the cAMP-unresponsive parathyroid hormone and glucagon genes and gave no transcriptional responses to 8-bromo-cAMP, nor could they produce specific gel-shift patterns or compete for binding to a labeled "active CRE." The first group consists of CRE sequences that contain the 8 bp palindrome 5'-TGACGTCA-3', flanked by several bases that are known to be permissive for both transcriptional activity and specific protein binding (Deutsch, P. J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988); Deutsch, P. J. et al., J. Bio. Chem. (in press)). The second group consists of the identical oligonucleotide duplexes in which the core 8 bp element was mutated by the deletion of a single base to form the phorbol ester-responsive sequence 5'-TGAGTCA-3'. Although structurally very similar to CREs, these sequences exhibit functional and binding properties that allow exclusion of recombinant phage expressing TRE-binding proteins and other proteins that may recognize the CRE/TRE motifs non-specifically. The final group corresponds to "inactive CRE's." These oligonucleotide duplexes contain the CRE motif 5'-TGACGTCA-3', but are flanked by the sequences that are not permissive for either cAMP stimulated gene transcription or specific protein binding to the CRE.

Using this strategy only recombinant phage that bind the active CREs and not the mutant TREs or the inactive CREs were considered to be true positives. Only two of the five recombinant phages initially identified fulfilled all of the binding criteria specific to the native CREB protein from JEG-3 human choriocarcinoma cells (FIG. 1A). Analysis of the cDNA inserts from these two phages indicated that they contained identical 2.4 kb DNA inserts and probably represent duplicates of the same phage.

The specific procedure for detection of a positive recombinant fusion protein in a λ gt11 expression library containing human placental cDNAs was as follows:

IPTG-induced proteins from plates containing plaque-purified recombinant phages were bound to nitrocellulose filters and probed separately as described (Singh, H. et al., Cell 52:415 (1988)) with radioactive duplex oligonucleotides containing either an octomeric cAMP response element (CRE) or heptomeric TPA response element (TRE). The CRE-containing probe, but not the TRE-containing probe, was specifically bound by the protein encoded by the recombinant phage. The TRE-containing probe was designed according to the sequence outlined by Angel, P. et al., Cell 49:729 (1987). Previously the element has been shown to be incapable of competing for specific binding to labeled CRE-containing probes in gel-shift assays (Deutsch, P. J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988)). The CRE probe differed from the TRE probe only by the additional C-G base-pair in parentheses, and was shown to impart a 15-30 fold stimulation of transcription in response to 8-bromo cAMP when placed upstream of a minimal promoter element.

5'-GATCCGGCTGAC(G)TCATCAAGCTA-3' CRE probe SEQ ID NO:1!

3'-GCCGACTG(C)AGTAGTTCGATCTAG-5' TRE probe SEQ ID NO:2!

The cDNA library was obtained from Clontech Laboratories, Inc., Palo Alto, Calif.

The radioactive CRE-containing probe used to select the recombinant phage will likewise bind to a protein present in a cell line of placental origin (JEG-3). Proteins in whole cell extracts of placental JEG-3 cells were separated by electrophoresis on a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was incubated with the radioactive CRE probe, revealing two intensely radioactive bands corresponding to proteins of apparent molecular weights of 38 Kd and 36 Kd.

The Southwestern blot analysis of DNA binding activity in extracts of placental JEG-3 cells was as follows:

To demonstrate that the radioactive CRE-containing probe, used to select the recombinant phage, also binds to the 38 kd CREB protein from JEG-3 cells when immobilized on nitrocellulose membranes, a Southwestern analysis was performed. 50 ug of extract was separated on 10% denaturing SDS gels and then electrotransferred to nitrocellulose membranes. The membranes were then exposed as described (Singh, H. et al., Cell 52:415 (1988)) to radioactive binding site probes containing either a CRE probe or TRE probe. The labeled CRE probe bound strongly to proteins of 38 Kd and 36 Kd, and weakly to a 26 Kd protein in these extracts after 24 h of autoradiography. The specific binding of this protein(s) to the CRE element is a finding that is consistent with our earlier observations using UV-crosslinking (Deutsch, P .J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988)). However, the TRE probe gave only weak signals even after five days of autoradiography. These differences in signal strengths may be a consequence of the relative abundances of the proteins which bind these elements, or to differences in the degree of renaturation achieved after transfer to nitrocellulose. Molecular weights of marker proteins are shown on the left of the autoradiograms.

The protein encoded by the beta-galactosidase fusion gene was analyzed by UV-crosslinking in the presence or absence of unlabeled competitor DNAs followed by transfer to nitrocellulose.

Ultraviolet light cross-linking of lysogen extracts was used to demonstrate the galactosidase fusion protein responsible for specific binding to the labeled CRE probe. UV-crosslinking was performed as described earlier (Deutsch, P. J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988)) using 50 ug of total protein from lysogen extracts from either the recombinant phage (center panel) or the negative control λ gt11 recombinant phage. The body-labeled, bromo-deoxyuridine incorporated probes were prepared by primed synthesis of the synthetic oligonucleotide 5'-AAAGCCAGAGGTGTCTGAC(G)TCATGCTTTATAACATCCTCTTGATTAG-CTA-3' SEQ ID NO:3! using the 15 base primer 5'-TAGCTAATCAAGAGG-3'. The G in parentheses represents the single base insertion in the CRE relative to the TRE. After separating bound proteins on10% SDS-gels, the proteins were transferred to nitrocellulose membranes and stained using anti-β-galactosidase antibodies. The major galactosidase species account for most of the specific binding. However, there were faint bands at lower molecular weights which presumably are due to binding to breakdown products of the apparent 137 Kd fusion protein, because negative control lysogens from the same Y1089 host cells showed no specific (or non-specific) binding to the CRE-containing probe. The specificity of binding to the CRE probe was confirmed by the lack of competition by the unlabeled TRE-containing probe.

Thus, the results confirmed that the B-galactosidase fusion protein was responsible for binding to the radioactive CRE-probe and that this binding is prevented in the presence of unlabeled CRE, but not unlabeled TRE, even at a 1000-fold molar excess.

Finally, to demonstrate that the fusion protein bound specifically to the CRE element in the context of a cellular promoter, a footprint analysis using the technique of digestion of DNA with exonuclease III was performed (Shalloway, D. et al., Cell 20:411 (1980)). The DNA construction comprised of the somatostatin CRE oligonucleotide duplex joined to the promoter sequence of the α-gonadotropin gene at position-100 (Deutsch, P. J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988)). The bacterial lysogen extract and extracts of JEG-3 cells provide similar protection of the CRE.

The exonuclease III protection footprinting procedure of the CRE by DNA binding activity in lysogen extracts of phage G1 is described as follows:

The radioactive probe used consisted of a CRE flanked by the native sequences found surrounding this element in the somatostatin gene linked to a 144 bp fragment of the α-gonadotropin gene promoter extending from -100 to +44 (Deutsch, P. J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988)). Both sense and antisense DNA strands were 5' end-labeled with ³² P, cut with a restriction endonuclease and the single end labeled DNAs were isolated by electrophoresis on 4% polyacrylamide gels. Binding reactions with lysogen and JEG-3 whole cell extracts were performed as described previously for gel shift assays (Deutsch, P. J. et al., Proc. Natl. Acad. Sci. USA 85:7922 (1988); Deutsch, P. J. et al., J. Bio. Chem. (in press)). The radioactive probes in the presence of cell extracts were then exposed to 100 units of Exonuclease III for 10 minutes at 37° C. The final radioactive products were analyzed by electrophoresis on 8% sequencing gels.

The amino acid sequence of 326 residues (m.w. 35,024) deduced from the nucleotide sequence of the subcloned cDNA shows several interesting structural features characteristic of DNA-binding transcription factors belonging to a new class recognized as leucine zipper proteins (Landschultz, W. H. et al., Science 240:1760 (1988)) as shown in FIG. 1. This class of proteins includes myc, fos, C/EBP, GCN4 and c-jun. Comparisons of leucine zipper regions in the structure of CREB and then other DNA binding proteins are shown in FIG. 3. A hypothetical "leucine zipper sequence" in which four leucines are spaced seven residues apart is located near the carboxyl terminus of the protein. The sequence was recently proposed by Landschultz, Johnson, and McKnight (Landschultz, W. H. et al., Science 240:1760 (1988)) to be a region involved in the formation of protein homodimers or other protein-protein interactions.

A computer search for sequence similarities between CREB and c-jun revealed a single region of 61% identity of amino acids (19 of 31 residues) between positions 270 to 300 of CREB and 254 to 284 of c-jun as shown in FIG. 4. These regions of similarity are located adjacent to the leucine zipper regions of the two proteins and constitute basic domains in which over 50% of the residues are either arginine or lysine. There is also a similarity of sequence in this region with GCN4, a protein previously noted to have similarity to c-jun (Bohmann, D. et al., Science 238:1386 (1987); and Angel, P. et al. Nature 332:166 (1988)). Without being bound by the theory, the similarities of sequences limited to this basic domain suggests that all these proteins bind to similar palindromic sequences; either TGACGTCA (CREB) or TGAGTCA (c-jun and GCN4). The high positive charge densities of these regions of the DNA binding proteins would be compatible with close contact with the negatively charged phosphate backbone of the DNA.

Although no additional regions of similarity were discerned for the primary sequence of CREB and c-jun, comparison of the predicted secondary structures shows several notable features as shown in FIG. 5. As expected, the zipper regions at the carboxyl terminus of the two proteins consist entirely of alpha helix (Landschultz, W. H. et al., Science 240:1760 (1988)). However, the remainder of the sequences located amino terminal to the basic domains of both proteins are predominantly random coil and are highly acidic. The sequences of CREB (residues 1-268) and c-jun (residues 1-225) have ratios of acidic to basic residues of 2.5 and 2.0, respectively. The sequence of CREB between residues 1 to 268 contains 25 glutamic acids and aspartic acids and 11 lysines and arginines. The corresponding sequence of c-jun between residues 1 to 225 contains 22 glutamic acids and aspartic acids and 11 lysines and arginines. These acidic regions of transcriptional proteins may be important activator regions for interactions with the basic transcriptional machinery and have been referred to as "acid blobs" or "negative noodles" to describe the conformationally poorly-defined structure of a polypeptide that can function almost irrespective of sequence provided that there are a sufficient number of acidic residues clustered or scattered about (Sigler, P. S., Nature 333:210 (1988); Hope, I. A. et al., Nature 333:635 (1988); Ma, J. et al., Cell 48:847 (1987); and Gill, G. et al., Cell 51:121 (1987)).

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 14     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     #                24TCAA GCTA     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 24 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     #                24GTCA GCCG     - (2) INFORMATION FOR SEQ ID NO:3:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 51 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     #             51TCTGACG TCATGCTTTA TAACATCCTC TTGATTAGCT A     - (2) INFORMATION FOR SEQ ID NO:4:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 15 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     #    15     - (2) INFORMATION FOR SEQ ID NO:5:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 2475 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 126..1106     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     - GAATTCGGGC GCGCCGGAGG TGTAGTTTGA CGCGGTGTGT TACGTGGGGG AG - #AGAATAAA       60     - ACTCCAGCGA GATCCGCGCC GTGAACGAAA GCAGTGACGG AGGAGCTTGT AC - #CACCGGTA      120     #CAG CAG AGT GGA CAT        167 GCC GAG AAC     #Glu Asn Gln Gln Ser Gly Hisy Ala     #     10     - GCA GCT GTA ACA GAA GCT GAA AAC CAA CAA AT - #G ACA GTT CAA GCC CAG      215     Ala Ala Val Thr Glu Ala Glu Asn Gln Gln Me - #t Thr Val Gln Ala Gln     # 30     - CCA CAG ATT GCC ACA TTA GCC CAG GTA TCT AT - #G CCA GCA GCT CAT GCA      263     Pro Gln Ile Ala Thr Leu Ala Gln Val Ser Me - #t Pro Ala Ala His Ala     #                 45     - ACA TCA TCT GCT CCC ACC GTA ACT CTA GTA CA - #G CTG CCC AAT GGG CAG      311     Thr Ser Ser Ala Pro Thr Val Thr Leu Val Gl - #n Leu Pro Asn Gly Gln     #             60     - ACA GTT CAA GTC CAT GGA GTC ATT CAG GCG GC - #C CAG CCA TCA GTT ATT      359     Thr Val Gln Val His Gly Val Ile Gln Ala Al - #a Gln Pro Ser Val Ile     #         75     - CAG TCT CCA CAA GTC CAA ACA GTT CAG ATT TC - #A ACT ATT GCA GAA AGT      407     Gln Ser Pro Gln Val Gln Thr Val Gln Ile Se - #r Thr Ile Ala Glu Ser     #     90     - GAA CAT TCA CAG GAG TCA GTG GAT AGT GTA AC - #T GAT TCC CAA AAC CGA      455     Glu His Ser Gln Glu Ser Val Asp Ser Val Th - #r Asp Ser Gln Asn Arg     #110     - AGG GAA ATT CTT TCA AGG AGG CCT TCC TAC AG - #G AAA ATT TTG AAT GAC      503     Arg Glu Ile Leu Ser Arg Arg Pro Ser Tyr Ar - #g Lys Ile Leu Asn Asp     #               125     - TTA TCT TCT GAT GCA CCA GGA GTG CCA AGG AT - #T GAA GAA GAG AAG TCT      551     Leu Ser Ser Asp Ala Pro Gly Val Pro Arg Il - #e Glu Glu Glu Lys Ser     #           140     - GAA GAG GAG ACT TCA GCA CCT GCC ATC ACC AC - #T GTA ACG GTG CCA ACT      599     Glu Glu Glu Thr Ser Ala Pro Ala Ile Thr Th - #r Val Thr Val Pro Thr     #       155     - CCA ATT TAC CAA ACT AGC AGT GGA CAG TAT AT - #T GCC ATT ACC CAG GGA      647     Pro Ile Tyr Gln Thr Ser Ser Gly Gln Tyr Il - #e Ala Ile Thr Gln Gly     #   170     - GGA GCA ATA CAG CTG GCT AAC AAT GGT ACC GA - #T GGG GTA CAG GGC CTG      695     Gly Ala Ile Gln Leu Ala Asn Asn Gly Thr As - #p Gly Val Gln Gly Leu     175                 1 - #80                 1 - #85                 1 -     #90     - CAA ACA TTA ACC ATG ACC AAT GCA GCA GCC AC - #T CAG CCG GGT ACT ACC      743     Gln Thr Leu Thr Met Thr Asn Ala Ala Ala Th - #r Gln Pro Gly Thr Thr     #               205     - ATT CTA CAG TAT GCA CAG ACC ACT GAT GGA CA - #G CAG ATC TTA GTG CCC      791     Ile Leu Gln Tyr Ala Gln Thr Thr Asp Gly Gl - #n Gln Ile Leu Val Pro     #           220     - ACC AAC CAA GTT GTT GTT CAA GCT GCC TCT GG - #A GAC GTA CAA ACA TAC      839     Thr Asn Gln Val Val Val Gln Ala Ala Ser Gl - #y Asp Val Gln Thr Tyr     #       235     - CAG ATT CGC ACA GCA CCC ACT AGC ACT ATT GC - #C CCT GGA GTT GTT ATG      887     Gln Ile Arg Thr Ala Pro Thr Ser Thr Ile Al - #a Pro Gly Val Val Met     #   250     - GCA TCC TCC CCA GCA CTT CCT ACA CAG CCT GC - #T GAA GAA GCA CCA CGA      935     Ala Ser Ser Pro Ala Leu Pro Thr Gln Pro Al - #a Glu Glu Ala Pro Arg     255                 2 - #60                 2 - #65                 2 -     #70     - AAG AGA GAG GTC CGT CTA ATG AAG AAC AGG GA - #A GCA GCT CGA GAG TGT      983     Lys Arg Glu Val Arg Leu Met Lys Asn Arg Gl - #u Ala Ala Arg Glu Cys     #               285     - CGT AGA AAG AAG AAA GAA TAT GTG AAA TGT TT - #A GAA AAC AGA GTG GCA     1031     Arg Arg Lys Lys Lys Glu Tyr Val Lys Cys Le - #u Glu Asn Arg Val Ala     #           300     - GTG CTT GAA AAT CAA AAC AAG ACA TTG ATT GA - #G GAG CTA AAA GCA CTT     1079     Val Leu Glu Asn Gln Asn Lys Thr Leu Ile Gl - #u Glu Leu Lys Ala Leu     #       315     - AAG GAC CTT TAC TGC CAC AAA TCA GAT TAATTTGGG - #A TTTAAATTTT     1126     Lys Asp Leu Tyr Cys His Lys Ser Asp     #   325     - CACCTGTTAA CGTGGAAAAT GGACTGGCTT GGCCACAACC TGAAAGACAA AA - #TAAACATT     1186     - TTATTTTCTA AACATTTCTT TTTTTCTATG CGCAAAACTG CCTGAAAGCA AC - #TACAGAAT     1246     - TTCATTCATT TGTGCTTTTG CATTAAACTG TGAATGTTCC AACACCTGCC TC - #CACTTCTC     1306     - CCCTCAAGAA ATTTTCAACG CCAGGAATCA TGAAGAGACT TCTGCTTTTC AA - #CCCCCACC     1366     - CTCCTCAAGA AGTAATAATT TGTTTACTTG TAAATTGATG GGAGAAATGA GG - #AAAAGAAA     1426     - ATCTTTTTAA AAATGATTTC AAGGTTTGTG CTGAGCTCCT TGATTGCCTT AG - #GGACAGAA     1486     - TTACCCCAGC CTCTTGAGCT GAAGTAATGT GTGGGCCGCA TGCATAAAGT AA - #GTAAGGTG     1546     - CAATGAAGAA GTGTTGATTG CCAAATTGAC ATGTTGTCAC ATTCTCATTG TG - #AATTATGT     1606     - AAAGTTGTTA AGAGACATAC CCTCTAAAAA AGAACTTTAG CATGGTATTG AA - #GGAATTAG     1666     - AAATGAATTT GCAGTGCTTT TTATGTATGT TGTCTTCTTC AATACTGAAA AT - #TTGTCCTT     1726     - GGTTCTTAAA AGCATTCTGT ACTAATACAG CTCTTCCATA GGGCAGTTGT TG - #CTTCTTAA     1786     - TTCAGTTCTG TATGTGTTCA ACATTTTTGA ATACATTAAA AGAAGTAACC AA - #CTGAACGA     1846     - CAAAGCATGG TATTTGAATT TTAAATTAAA GCAAAGTAAA TAAAAGTACA AA - #GCATATTT     1906     - TAGTTAGTAC TAAATTCTTA GTAAAATGCT GATCAGTAAA CCAATCCCTT GA - #GTTATATA     1966     - ACAAGATTTT TAAATAAATG TTATTGTCCT CACCTTCAAA AATATTTATA TT - #GTCACTCA     2026     - TTTACGTAAA AAGATATTTC TAATTTACTG TTGCCCATTG CACTTACATA CC - #ACCACCAA     2086     - GAAAGCCTTC AAGATGTCAA ATAAAGCAAA GTGATATATA TTTGTTTATG AA - #ATGTTACA     2146     - TGTAGAAAAA TACTGATTTT AAATATTTTC CATATTAACA ATTTAACAGA GA - #ATCTCTAG     2206     - TGAATTTTTT AAATGAAAGA AGTTGTAAGG ATATAAAAAG TACAGTGTTA GA - #TGTGCACA     2266     - AGGAAAGTTA TTTTCAGACA TATTTGAATG ACTGCTGTAC TGCAATATTT GG - #ATTGTCAT     2326     - TCTTACAAAA CATTTTTTTG TTCTCTTCTA AAAACACTAG TTATTAGTTC TG - #CTTTAGCT     2386     - TTCCAATATG CTGTATAGCC TTTGTCATTT TATAATTTTA ATTCCTGATT AA - #AACAGTCT     2446     #          2475    TCCC CCCGAATTC     - (2) INFORMATION FOR SEQ ID NO:6:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 327 amino               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     - Met Thr Met Glu Ser Gly Ala Glu Asn Gln Gl - #n Ser Gly His Ala Ala     #                 15     - Val Thr Glu Ala Glu Asn Gln Gln Met Thr Va - #l Gln Ala Gln Pro Gln     #             30     - Ile Ala Thr Leu Ala Gln Val Ser Met Pro Al - #a Ala His Ala Thr Ser     #         45     - Ser Ala Pro Thr Val Thr Leu Val Gln Leu Pr - #o Asn Gly Gln Thr Val     #     60     - Gln Val His Gly Val Ile Gln Ala Ala Gln Pr - #o Ser Val Ile Gln Ser     # 80     - Pro Gln Val Gln Thr Val Gln Ile Ser Thr Il - #e Ala Glu Ser Glu His     #                 95     - Ser Gln Glu Ser Val Asp Ser Val Thr Asp Se - #r Gln Asn Arg Arg Glu     #           110     - Ile Leu Ser Arg Arg Pro Ser Tyr Arg Lys Il - #e Leu Asn Asp Leu Ser     #       125     - Ser Asp Ala Pro Gly Val Pro Arg Ile Glu Gl - #u Glu Lys Ser Glu Glu     #   140     - Glu Thr Ser Ala Pro Ala Ile Thr Thr Val Th - #r Val Pro Thr Pro Ile     145                 1 - #50                 1 - #55                 1 -     #60     - Tyr Gln Thr Ser Ser Gly Gln Tyr Ile Ala Il - #e Thr Gln Gly Gly Ala     #               175     - Ile Gln Leu Ala Asn Asn Gly Thr Asp Gly Va - #l Gln Gly Leu Gln Thr     #           190     - Leu Thr Met Thr Asn Ala Ala Ala Thr Gln Pr - #o Gly Thr Thr Ile Leu     #       205     - Gln Tyr Ala Gln Thr Thr Asp Gly Gln Gln Il - #e Leu Val Pro Thr Asn     #   220     - Gln Val Val Val Gln Ala Ala Ser Gly Asp Va - #l Gln Thr Tyr Gln Ile     225                 2 - #30                 2 - #35                 2 -     #40     - Arg Thr Ala Pro Thr Ser Thr Ile Ala Pro Gl - #y Val Val Met Ala Ser     #               255     - Ser Pro Ala Leu Pro Thr Gln Pro Ala Glu Gl - #u Ala Pro Arg Lys Arg     #           270     - Glu Val Arg Leu Met Lys Asn Arg Glu Ala Al - #a Arg Glu Cys Arg Arg     #       285     - Lys Lys Lys Glu Tyr Val Lys Cys Leu Glu As - #n Arg Val Ala Val Leu     #   300     - Glu Asn Gln Asn Lys Thr Leu Ile Glu Glu Le - #u Lys Ala Leu Lys Asp     305                 3 - #10                 3 - #15                 3 -     #20     - Leu Tyr Cys His Lys Ser Asp                     325     - (2) INFORMATION FOR SEQ ID NO:7:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 27 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     - Leu Thr Ser Asp Asn Asp Arg Leu Arg Lys Ar - #g Val Glu Gln Leu Ser     #                15     - Arg Glu Leu Asp Thr Leu Arg Gly Ile Phe Ar - #g     #            25     - (2) INFORMATION FOR SEQ ID NO:8:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 27 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     - Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gl - #n Asn Ser Glu Leu Ala     #                15     - Ser Thr Ala Asn Met Leu Arg Glu Gln Val Al - #a     #            25     - (2) INFORMATION FOR SEQ ID NO:9:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 27 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     - Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Ly - #s Asn Tyr His Leu Glu     #                15     - Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gl - #y     #            25     - (2) INFORMATION FOR SEQ ID NO:10:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 27 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     - Leu Gln Ala Glu Thr Asp Gln Leu Glu Asp Ly - #s Lys Ser Ala Leu Gln     #                15     - Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Gl - #u     #            25     - (2) INFORMATION FOR SEQ ID NO:11:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 27 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     - Leu Gln Ala Glu Glu His Gln Leu Leu Leu Gl - #u Lys Glu Lys Leu Gln     #                15     - Ala Arg Gln Gln Gln Leu Leu Lys Lys Ile Gl - #u     #            25     - (2) INFORMATION FOR SEQ ID NO:12:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 27 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     - Leu Val Gly Ala Glu Lys Arg Met Ala Thr Gl - #u Lys Arg Gln Leu Arg     #                15     - Cys Arg Gln Gln Gln Leu Gln Lys Arg Ile Al - #a     #            25     - (2) INFORMATION FOR SEQ ID NO:13:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 27 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     - Leu Thr Ser Glu Lys Asp Leu Leu Arg Lys Ar - #g Arg Glu Gln Leu Lys     #                15     - His Lys Leu Glu Gln Leu Arg Asn Ser Gly Al - #a     #            25     - (2) INFORMATION FOR SEQ ID NO:14:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 60 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: both               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     - Ile Asp Met Glu Ser Gln Glu Arg Ile Lys Al - #a Glu Arg Lys Arg Met     #                15     - Arg Asn Arg Ile Ala Ala Ser Lys Cys Arg Ly - #s Arg Lys Leu Glu Arg     #            30     - Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Le - #u Lys Ala Gln Asn Ser     #        45     - Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Gl - #u Gln     #    60     __________________________________________________________________________ 

What is claimed is:
 1. A substantially purified DNA encoding cAMP-responsive transcription enhancer binding protein (CREB), said CREB having the amino acid sequence shown in FIG.
 1. 2. Isolated DNA encoding CRE recognition or binding fragments of the protein encoding by the DNA of claim
 1. 3. A construct comprising the DNA of claim 1 or the DNA of claim 2 encoding CRE recognition or binding fragments.
 4. A construct comprising a promoter operably-linked to a heterologous gene coding for a protein or polypeptide and comprising the CRE palindrome with the nucleotide sequence TGACGTCA and the DNA of claim 1 wherein said CRE palindrome is operably-linked to said promoter.
 5. A host cell transformed with the DNA of claim
 1. 6. A method of increasing production of a heterologous protein or polypeptide, said method comprising:(a) transforming a eukaryotic host cell with a construct comprising(i) a minimum selectable region comprising a eukaryotic promoter operably-linked to a heterologous gene; (ii) a minimum selectable region comprising the CRE palindrome with the nucleotide sequence TGACGTCA operably linked to said promoter of said heterologous gene; (iii) a minimum selectable region comprising a eukaryotic promoter operably-linked to the DNA of claim 1; (b) culturing said transformed eukaryotic host cell under conditions selected for favorable growth of said host cell; and (c) adding cAMP to said host cell to stimulate said heterologous gene to express the encoded protein or polypeptide.
 7. A method of increasing production of a heterologous protein or polypeptide, said method comprising:(a) transforming a eukaryotic host cell with a construct comprising(i) a minimum selectable region comprising a eukaryotic promoter operably-linked to a transcription unit; (ii) a minimum selectable region comprising the CRE palindrome with the nucleotide sequence TGACGTCA operably linked to said promoter of said transcription unit; (b) transforming said eukaryotic cell with a construct comprising(i) a minimum selectable region comprising a eukaryotic promoter operably-linked to the DNA of claim 1; (c) culturing said transformed eukaryotic host cell under conditions selected for favorable growth of said host cell; and (d) adding cAMP to said host cell to stimulate said heterologous gene to express the encoded protein or polypeptide.
 8. A method for increasing transcription of DNA, said method comprising:(a) transforming a eukaryotic host cell with a construct comprising(i) a minimum selectable region comprising a eukaryotic promoter operably-linked to a heterologous gene; (ii) a minimum selectable region comprising the CRE palindrome with the nucleotide sequence TGACGTCA operably linked to said promoters of said heterologous gene; (iii) a minimum selectable region comprising a eukaryotic promoter operably-linked to the DNA of claim 1; (b) culturing said transformed eukaryotic host cell under conditions selected for favorable growth of said host cell; and (c) adding cAMP to said host cell to stimulate said heterologous gene to stimulate transcription of said heterologous gene.
 9. A method for increasing transcription of DNA, said method comprising:transforming a eukaryotic host cell with a construct comprising(i) a minimum selectable region comprising a eukaryotic promoter operably-linked to a transcription unit; (ii) a minimum selectable region comprising the CRE palindrome with the nucleotide sequence TGACGTCA operably linked to said promoter of said transcription unit; (b) transforming said eukaryotic cell with a construct comprising(i) a minimum selectable region comprising a eukaryotic promoter operably-linked to the DNA of claim 1; (c) culturing said transformed eukaryotic host cell under conditions selected for favorable growth of said host cell; and(d) adding cAMP to said host cell to stimulate transcription of said transcription unit.
 10. The method of claim 6, 7, 8 or 9 wherein the promoter operably-linked to the heterologous gene is the same sequence as the promoter operably-linked to said DNA of claim
 1. 11. The method of claim 6, 7, 8 or 9 wherein the promoter operably-linked to the transcription unit uses RNA Polymerase II.
 12. The method of claim 6, 7, 8 or 9 wherein the transcription unit codes for a transposase.
 13. The method of claim 8 or 9 wherein the heterologous gene transcribes an anti-sense RNA.
 14. A method for decreasing expression of a protein comprising:(a) transforming a eukaryotic host cell with the construct of claim 8 or 9 wherein said transcription unit transcribes the sequence of an anti-sense RNA; (b) culturing said transformed eukaryotic host cell under conditions selected for favorable growth of said host cell; (c) adding camp to said host cell to stimulate transcription of said anti-sense RNA; and (d) culturing said host cell under conditions favorable to hybridization of said anti-sense RNA to said mRNA whereby expression of said protein is decreased.
 15. The method of claim 8 or 9, wherein said construct is integrated into the host's genome.
 16. A construct comprising a promoter operably-linked to a heterologous gene coding for a protein or polypeptide and comprising the CRE palindrome with the nucleotide sequence TGACGTCA and nucleotides encoding a CRE recognition or binding fragment of the protein encoded by the DNA of claim 2 wherein said CRE palindrome is operably-linked to said promoter.
 17. A host cell transformed with the DNA of claim
 2. 18. The method of claim 14, wherein said mRNA is homologous to said host cell.
 19. The method of claim 14, wherein said mRNA is heterologous to said host cell.
 20. The method of claim 14 wherein said construct is integrated into the host's genome. 